The invention relates to devices for separating magnetic or magnetizable particles from liquids by a magnetic field produced by one or more permanent magnets.
The invention further relates to methods for separating magnetic or magnetizable particles from liquids by a magnetic field produced by one or more permanent magnets. The devices and methods can be used, for example, for applications in biochemistry, molecular genetics, microbiology, medical diagnostics and forensic medicine.
Methods based on magnetic separation using specifically binding, magnetically attractable particles are increasingly gaining in significance in the field of sample preparation for diagnostic or analytic examinations. This is true, in particular, for automated processes, since it is thereby possible to analyse a large number of samples within a short period of time and to dispense with labor-intensive centrifugation steps. This creates the conditions required for efficient screening at a high sample throughput. This is extremely important for applications in molecular-genetic studies or in the field of medical diagnostics, for example, as it is practically impossible to cope with very large numbers of samples by purely manual handling. Further important fields of application relate to pharmaceutical screening methods for identification of potential pharmaceutical active agents.
The basic principle of magnetic separation of substances from complex mixtures is based on the process of functionalizing magnetic particles (magnetizable or magnetically attractable particles) in a specific manner for the intended separation process. That is, they are provided, by chemical treatment, with specific binding properties for the target substances to be separated. The size of these magnetic particles is typically in the range of approx. 0.05 to 500 μm.
Magnetic particles that have specific binding properties for certain substances and can be used to remove these substances from complex mixtures are described, for example, in German published patent application DE 195 28 029 A1 and are commercially available, e.g., from chemagen Biopolymer-Technologie AG, DE-52499 Baesweiler, Germany.
In known separating methods the functionalized magnetic particles are added in a first step (“binding step”) to a mixture to be purified which contains the target substance(s) in a liquid promoting the binding of the target substance molecules to the magnetic particles (binding buffer). This causes a selective binding of the target substance(s) present in the mixture to the magnetic particles. Subsequently, these magnetic particles are immobilized on a site of the interior wall of the reaction vessel by employing magnetic forces, that is, a magnetic field, for instance by a permanent magnet (“pellet”). Thereafter, the liquid supernatant is separated and discarded, for example by suction or decanting. Since the magnetic particles are immobilized in the manner described, it is largely prevented that these particles are separated along with the supernatant.
Subsequently, the immobilized magnetic particles are again re-suspended. For this purpose, an eluting liquid or eluting buffer is used that is suitable for breaking the bond between the target substance(s) and the magnetic particles, so that the target substance molecules can be released from the magnetic particles and separated along with the elution liquid, while the magnetic particles are immobilized by the action of the magnetic field. One or more washing steps may be carried out prior to the elution step.
Devices of various types have been described for carrying out separation processes by magnetic particles. German utility model DE 296 14 623 U1 discloses a magnetic separator provided with movable permanent magnets. As an alternative, it is proposed to move the reaction vessel containing the magnetic particles by mechanical drive, relative to a fixedly mounted permanent magnet. The device described in German published patent application DE 100 63 984 A1, which is provided with a magnetic holder and a movable reaction vessel holder, works according to a similar principle.
By using the above-mentioned devices, it is possible to immobilize or accumulate the magnetic particles on the interior wall or on the bottom of the reaction vessel as a “pellet”. These devices are, however, not suitable for removing the magnetic particles from a reaction vessel. As a consequence, it is necessary to exhaust the liquid from each individual reaction vessel by suction in order to separate the liquid from the magnetic particles. This is a disadvantage, as it entails high material consumption (disposable pipette tips). Furthermore, it is unavoidable that individual magnetic particles are also sucked off, thus leading to a high error rate. Other errors can be caused by liquids dripping down, leading to cross-contamination.
German Patent DE 100 57 396 C1 proposes a magnetic separator provided with a plurality of rotatable bars that can be magnetized by an electromagnetic excitation coil. By immersing the bar in the liquid containing magnetic particles and withdrawing the bar in the magnetized state, the magnetic particles can be removed from the liquid and, if required, transferred to another reaction vessel where they can be re-released into a liquid, e.g. a wash or elution liquid, by deactivating the excitation coil.
A disadvantage of this device is that the magnetic field produced by the excitation coil is not sufficiently homogenous so that the individual bars, depending on their position within the ring-shaped excitation coil, are magnetized to a different extent. This disadvantage is particularly eminent where a large number of bars is required. In addition, the excitation coil requires a relatively large space, which results in constructional limitations.
Above all, the known devices are not suitable for simultaneous treatment of large numbers of samples as is required in high-throughput applications (e.g., microtiter plates with 364 or 1536 wells).